HLA-DR-TYPING, DQ-TYPING AND DP-TYPING USING PCR AMPLIFICATION AND IMMOBILIZED PROBES
HLA-DR-TYPING, DQ-TYPING AND DP-TYPING USING PCR AMPLIFICATION AND IMMOBILIZED PROBES
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DOI：
10.1111/j.1744-313x.1991.tb00005.x
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发表时间：
1991-02-01
期刊：
影响因子：
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通讯作者：
WALSH, PS
中科院分区：
文献类型：
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作者：
ERLICH, H;BUGAWAN, T;WALSH, PS
一种简便、快速、准确的HLA Ⅱ类基因多态性分型方法，在疾病易感性、组织移植、个体识别和人类遗传学等领域具有重要的应用价值。 在这里，我们描述了一种基于聚合酶链反应（PCR）扩增和与寡核苷酸探针杂交的分析II类序列多态性的方法。 基于序列的HLA分型策略（如寡核苷酸探针杂交）的一个有价值的特性是，它们揭示了两个等位基因如何以及在何处不同，而不仅仅是它们可以在操作上区分。 HLA多态性的性质和位置在疾病相关性研究中似乎是至关重要的，并且在移植的组织分型中可能是重要的。 随着这项技术应用于越来越多的样本，特别是非高加索人种，DRB 1、DPB 1和DQB 1基因座的新等位基因很可能被鉴定出来。 一个新的等位基因被发现作为一个不寻常的模式的探针结合，然后通过测序确认。 观察到这种模式是因为II类多态性定位于特定区域，并且几乎所有"新"等位基因在探针结合区域具有多态性。 显然，在没有分型探针的区域中具有新的多态性序列的任何新等位基因都不能通过寡核苷酸分型来揭示。使用这里描述的PCR引物和探针，区分了7个DQA 1等位基因、15个DQB 1等位基因、18个DPB 1等位基因和32个DRB 1等位基因。 当然，额外的引物和/或探针可以增加寡核苷酸斑点印迹分型的等位基因区分。 这些辣根过氧化物酶（HRP）标记的寡核苷酸探针是稳定的（在4 ℃下储存时> 2年），分型系统简单而耐用。 来自CEPH家系的500多个样本（未发表的数据; A. B。Begovich等人，手稿的准备）和> 1000无关的样本已经打字通过这一程序。 虽然这种斑点印迹/寡核苷酸杂交程序是HLA II类分型的强大而精确的方法，但随着分析所需探针数量的增加，程序的复杂性也会增加。 反向斑点印迹法基于固定化探针阵列，允许在一个单一的杂交反应中对单个样品进行分型。 在这种方法中，将一组未标记的寡核苷酸固定在尼龙膜上。 PCR产物在扩增反应期间通过使用生物素化的引物进行标记并与膜杂交。 使用链霉亲和素-HRP缀合物和显色或荧光底物检测与给定探针特异性杂交的结合PCR产物的存在。 该方法为临床样本的HLA分型提供了最简单、最快速的方法。
A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing.With the PCR primers and probes described here, 7 DQA1 alleles, * 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (> 2 years when stored at 4-degrees-C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and > 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates. This method offers the simplest and most rapid approach to the HLA typing of clinical samples.